SARS-CoV-2 Subgenomic RNA Kinetics in Longitudinal Clinical Samples

Background Given the persistence of viral RNA in clinically recovered coronavirus disease 2019 (COVID-19) patients, subgenomic RNAs (sgRNAs) have been reported as potential molecular viability markers for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, few data are available on their longitudinal kinetics, compared with genomic RNA (gRNA), in clinical samples. Methods We analyzed 536 samples from 205 patients with COVID-19 from placebo-controlled, outpatient trials of peginterferon Lambda-1a (Lambda; n = 177) and favipiravir (n = 359). Nasal swabs were collected at 3 time points in the Lambda (days 1, 4, and 6) and favipiravir (days 1, 5, and 10) trials. N-gene gRNA and sgRNA were quantified by quantitative reverse transcription polymerase chain reaction. To investigate the decay kinetics in vitro, we measured gRNA and sgRNA in A549 ACE2+ cells infected with SARS-CoV-2, following treatment with remdesivir or dimethylsulfoxide control. Results At 6 days in the Lambda trial and 10 days in the favipiravir trial, sgRNA remained detectable in 51.6% (32/62) and 49.5% (51/106) of the samples, respectively. Cycle threshold (Ct) values for gRNA and sgRNA were highly linearly correlated (marginal R 2 = 0.83), and the rate of increase did not differ significantly in the Lambda trial (1.36 cycles/d vs 1.36 cycles/d; P  = .97) or the favipiravir trial (1.03 cycles/d vs 0.94 cycles/d; P  = .26). From samples collected 15–21 days after symptom onset, sgRNA was detectable in 48.1% (40/83) of participants. In SARS-CoV-2-infected A549 ACE2+ cells treated with remdesivir, the rate of Ct increase did not differ between gRNA and sgRNA. Conclusions In clinical samples and in vitro, sgRNA was highly correlated with gRNA and did not demonstrate different decay patterns to support its application as a viability marker.